NaCl/MeJA And MeJA-Ca²⁺ Regulate Physiological Metabolism And Resveratrol Enrichment During Peanut Germination

Resveratrol is a class of secondary metabolites formed by phenylpropane metabolism in higher plants,which has various physiological functions such as preventing cardiovascular disease,anti-cancer and antioxidant.Peanuts(Arachis hypogaea L.)are one of the natural sources of resveratrol and are rich in protein,oil and carbohydrates.After peanut germination,anti-nutritional factors are reduced,small molecule nutrients are increased,and resveratrol is accumulated.The study found that the growth of peanut sprouts was inhibited under abiotic stress but activated the key enzyme activities of resveratrol synthesis,which led to further accumulation of resveratrol.Ca2+ plays an important role as a second messenger of intercellular signal transduction in plant resistance to abiotic stress,regulation of plant growth,and in regulating the synthesis of secondary metabolites.In this paper,we investigated the mechanism of NaCl/MeJA and MeJA-Ca2+to regulate the physiology and resveratrol enrichment during peanut germination at the physiological metabolism,enzyme level and gene transcription level.The main findings are as follows:1.The effects of NaCl/MeJA treatment on the physiological metabolism and resveratrol synthesis in peanut sprouts were investigated.Under NaCl/MeJA stress,MDA content was significantly increased(p<0.05),ROS accumulated,and peanut sprout growth was inhibited;On the 5th day of germination,NaCl stress significantly increased POD and CAT activities,while MeJA stress significantly increased POD,SOD and CAT activities;NaCl significantly increased 4CL activity in cotyledons,and PAL and C4H activities in non-cotyledons;MeJA significantly increased C4H and 4CL activities in cotyledons,and PAL,C4H and 4CL activities in non-cotyledons(p<0.05).Resveratrol content under NaCl and MeJA treatments was 1.22 and 1.26 times higher than the control,and 2.62 and 2.82 times higher than the that in the seeds on the 5th day of germination,respectively.The increased antioxidant enzyme activities and resveratrol synthesis contributed to the alleviation of NaCl/MeJA stress.2.The effects of NaCl/MeJA treatment on the expression levels of resveratrol synthesis related enzyme genes and physiologically related genes during peanut germination were investigated.Under NaCl stress,PAL,C4H,4CL,and RS expression levels in cotyledons,and RS and 4 CL expression levels in non-cotyledons of peanut sprouts increased significantly(p<0.05).MeJA significantly increased PAL and 4CL expression levels in cotyledons(p<0.05),and 4CL and RS expression levels in non-cotyledons.NaCl/MeJA stress significantly enhanced POD,NAC65,and ERF6 expression levels in cotyledons,and POD,CAT,NAC65,ERF6,and DREB1 expression levels in non-cotyledons on the 5th day of germination(p<0.05).Antioxidant enzyme genes as well as physiologically related genes were upregulated to enhance the plant’s resistance to NaCl/MeJA stress.3.The regulatory effects of Ca2+on peanut sprout physiology and resveratrol accumulation under MeJA treatment were investigated.Under MeJA plus CaCl2 treatment,POD and SOD activities,and ferric ion reduction capacity were significantly increased,while MDA and H2O2 content were significantly decreased(p<0.05),thus alleviating MeJA stress.Sprout length and fresh weight were significantly enhanced(p<0.05).MeJA plus CaCl2 treatment significantly increased PAL and 4CL activities in cotyledons,PAL,C4H and 4CL activities in non-cotyledons,and enriched resveratrol.Ca2+ content,CaM content,and Ca2+ATPase activity were significantly increased by MeJA plus CaCl2 treatment In contrast,MeJA plus EGTA/LaCl3 inhibited the the above processes.These indicated that Ca2+ was involved in the synthesis of resveratrol and the enhancement of antioxidant system in peanut sprouts under MeJA stress,and acted as a downstream of MeJA.4.The effect of MeJA-Ca2+on the expression levels of resveratrol synthesis related enzyme genes,calcium binding protein genes and antioxidant enzyme genes in peanut sprouts on the 5th day of germination was investigated.MeJA plus CaCl2 treatment significantly upregulated PAL,C4H,RS,CaM and CDPK expression levels in both organs(p<0.05).The application of EGTA and LaCl3 reversed these effects.It suggested that Ca2+promoted intracellular Ca2+ signaling through up-regulation of CaM and CDPK expression,which in turn activated the expression of key enzyme genes for resveratrol synthesis and promoted resveratrol accumulation.The expression levels of SOD and CAT in cotyledons,and POD and SOD in non-cotyledons were significantly up-regulated by MeJA plus CaCl2 treatment(p<0.05).MeJA plus EGTA or LaCl3 inhibited the regulatory role of Ca2+on antioxidant enzymes and inhibited the growth of peanut sprouts.